Mechanisms of Allergic Immunity

Mechanisms of Allergic Immunity

Normal larynx Laryngeal oedema

Cellular culprits of allergy: Mast cells
Most informative early analysis conducted in patients with asthma Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity Provoked by allergenic and non allergenic substances Explained atopic and non-atopic asthma Explained why mast cell stabilising drugs worked

Cellular culprits of allergy: Mast cells??
Corticosteroid treatment worked, but had no effect on histamine release Anti-histamine treatment had little effect on asthma Could not explain ‘organ specificity’ of asthma Could not explain the hyperresponsive airway in asymptomatic asthmatics Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present)

Cellular culprits of allergy: T cells
The early evidence: Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics Activated T cells elevated in the peripheral blood of severe acute asthmatics Activated T cells in peripheral blood correlated with airway narrowing Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL T cells that release IL-5 co-localise with eosinophils Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction IL-5 promotes differentiation and regulates the survival of eosinophils Steroid treatment associated with a decrease in IL-5 producing cells

Cellular culprits of allergy: T cells
Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF- Inhibits macrophage activation Th2

Lebman & Coffman 1988 J Exp Med 168, 853-862

‘Textbook’ scheme of allergic immunity is centred around polarised Th cells
Ig isotype switch B IgE Th2 Th1 -ve MF -ve Eosinophil Differentiation and development Mast cell Where do Th2 cells come from? Why are they so dominant in allergic individuals? What are they really for?

The discovery of Th1 and Th2 subsets
Journal of Immunology 136,

In vitro - Th1 and Th2 subsets
IL-4 IFN-g T cell clones that make IFN-g, but not IL-4 T cell clones that make IL-4, but not IFN-g Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells In vitro - Th1 and Th2 subsets

Relevance in vivo - Infection
Non-healing BALB/c Resistant C57BL/6 Draining LN T cells express IL-4 mRNA Draining LN T cells express IFN-g mRNA T Leishmania - specific T cells Irradiated BALB/c recipient Resistance Reiner & Locksley Annu. Rev. Immunol. 13, , 1995

Relevance in vivo - Infection
IFN- / IL-12 or anti-IL-4 Pro-Th1 treatments or anti-Th2 treatments protect against infection

Leishmania resistance - mechanism
Macrophage and Leishmania Inflammatory Th1 T cell Th1 IFN- Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells

Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy
Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Th2 Th1

Tuberculoid leprosy

Lepromatous Leprosy

‘Textbook’ scheme of allergic immunity is centred around polarised Th cells
Immunological fashions 1960’s & 1970’s Immunoglobulin E 1970’s & 1980’s Mast cells & Eosinophils 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells 1990’s & 2000’s Microbial experience, Epithelium, Tregs Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy Allergy is a disease of impaired immune regulation Where is the regulatory lesion?

Allergic immune responses are much like any other immune
response and involves the same regulators Non self protein from allergen or pathogen Barrier: Skin, gut, lung, eye, nose etc Inflammation inc. MIP-1a, MCP-1 MIP-1b Activation and migration of dendritic cells to site of inflammation

Tracheal Dendritic Cells Langerhan’s cells
In-vitro differentiated monocyte-derived Dendritic Cell

Migration of immature DC to sites of inflammation
Sallusto et al., Eur. J. Immunol Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1-b which bind to, and trigger CCR1, CCR2 and CCR5 respectively. [Ca2+]i Time (s) Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i [Ca2+]i Time (s)

Migration of mature DC to 2º lymphoid tissue
Sallusto et al., Eur. J. Immunol Time (s) [Ca2+]i Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-b Mature DC respond to the lymph node derived CCR7 ligand MIP-3b Time (s) [Ca2+]i Time (s) [Ca2+]i

DC – T cell interactions in the lymph node
Mempel, T.R et al Nature 427: , 2004. Anti OVA TcR transgenic mouse Splenic DC Pulsed with Ag OVA T cells labelled GREEN DC labelled RED Not pulsed with Ag 0hr -18hr 2hr Anti-L selectin Ab Imaging at various timepoints

Early entry of DC to the lymph node Mempel, T
Early entry of DC to the lymph node Mempel, T.R et al Nature 427: , 2004. 1. DCs strategically cluster around HEV 18hr after entering the LN 2. Distribution of Ag-loaded DCs and T cells is ordered 4-5hr after T cells are injected

3. DC become highly migratory & change shape (20hr)

4. T cells cover large territories in LN

6. Short, serial T cell-DC contacts of ~ 5 minutes (2-4hrs after injection of T cells)
7. Stable T cell-DC conjugates of minutes (8-12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells

T cells start to proliferate and produce cytokines 44hr after transfer
5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again

More information than is provided by the antigen is exchanged between the DC and T cell
DC have a profound influence on the properties of the T cell that develops

DC Th Signals 1, 2 and 3 Signal 3 - pathogen polarised DC
Signal 1 antigen & antigen receptor Signal 3 - pathogen polarised DC Signal 2 B7 - CD28 Costimulation Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2?

DC Th Polarised DC subsets Signal 1 Signal 2 Signal 3
Integration of signals from pathogen/allergen and the extracellular milieu polarise the DC to produce qualitatively different signals 3 Signal 3 Th polarising signal The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells

Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216
Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pathogen-associated molecular patterns (PAMPS) Conserved microbial molecules shared by many pathogens Include: Bacterial lipopolysaccharides Peptidoglycan Zymosan Flagellin Unmethylated CpG DNA Pattern Recognition Receptors (PRR) Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins

Type 1 and 2 DC Polarising PAMPS
+ Class II CD40 + Type 1 PAMPS bind to PRR CD80/CD86 Type 2 PAMPS bind to PRR Th1 polarising factor IL-12 Th2 polarising factor CCL2 (MCP-1)

Type 1 PAMPS and their PRR
Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. Cruzi) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein Unmethylated CpG DNA dsDNA TLR 2 TLR 1 TLR 6 TLR 3 CD14 TLR 4 MD-2 TLR 9 Low level IL-12p70 Some ligands induce IL-10 or IL-12p35 High IL-12p70 IFN-a High IL-12p70 High IL-12p70 IFN-a

Type 2 PAMPS and their PRR
?

Endogenous molecular patterns
Include: Heat shock proteins (HSP60 HSP70 GP96) Extracellular matrix proteins (hyaluronan, fibronectin, fibrinogen) Immune complexes Surfactant protein A Necrotic cell components Pattern Recognition Receptors (PRR) Toll like receptors Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins

Indirect activation of DC by ‘modulatory tissue factors’
Direct activation by PAMP-PRR interactions Allergen Necrotic/apoptotic cell death - neo expression of PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components Activates the expression of costimulatory molecules on DC

DC polarisation by modulatory tissue factors
DC polarising factors IFN-g IFN-a IFN-b Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-b IL-18 DC polarising factors CCL7 (MCP-3), CCL13 (MCP-4), PGE2, Histamine Th0 to Th2 polarising cytokines CCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-b IL-18 Could be argued that the development of Th2 cells is the default pathway

Sources of modulatory tissue factors
Viruses Fungi Parasites Bacteria NK Epithelium Mast Fibroblast PGE2 CCR2L Histamine IFN-g IFN-a IL-18 Th2 Th1

Explains how Th2 arise, but…
…does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances? The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families

Neonatal & infant immune systems
Serial infections Age Immune response Th1 Th2 Th2 The intrauterine environment is powerfully Th2 – this imprints Th2 dominance upon the neonate Balanced Th1/Th2 at ~2yr

Delayed maturation of Th1 capacity
Few serial infections – hygiene, small family size etc Age Immune response Th1 Th2 Longer period of time in which to make and establish Th2 responses to environmental antigens (i.e. allergens) Unbalanced Th1/Th2 Th2 dominance at ~2yr

Do infections only reduce Th2 dominance by inducing Th1 responses?
Vaccinate with mycobacteria Aerosolised ovalbumin (OVA) Wheeze Wheeze OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE No asthma-like symptoms Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms?

Do infections only reduce Th2 dominance by inducing Th1 responses?
Vaccinate with mycobacteria Th CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGFb and IL-10 Wheeze No asthma-like symptoms Mycobacteria induced REGULATORY T cells

Th cell polarisation DC mediated – decision influenced by infection
Extracellular milieu - mediated

Priming conditions IFNg U/ml IL-4 pg/ml
Journal of Immunology 1 10 Factor increase over control 1 10 Factor increase over control Priming conditions IFNg U/ml IL-4 pg/ml Control Ab Anti-IFNg Ab IL-4 + control Ab IL-4 + anti-IFNg Ab

IL-4 from the innate immune system
IL-4 is not only a product of Th2 cells Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a Resting Mast cell IL-4, IL-5

Journal of Experimental Medicine, 1992 176 1381-1386
Sequential 2mm sections from a mucosal biopsy of a patient with asthma Tryptase IL-4 Journal of Experimental Medicine,

What properties and characteristics make a substance an allergen?
How do these properties disregulate the processes described?

L. destructor D. pteronyssinus D. pteronyssinus G. domesticus T. putrescentiae A. siro

Allergens of Dermatophagoides pteronyssinus
Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases)

Protease allergens can breach epithelial barriers
Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen

Proteases as activators of cells
Inactivators Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ?

Journal of Immunology 2001 167 1014-1021
PAR are also involved in: Induction of of epithelial cell & fibroblast proliferation Induction of cytokines & chemokine expression Induction of pharmacological mediator release Induction of metalloproteases Regulation of smooth muscle tone

Do protease allergens induce IL-4 release by Mast cells
Degranulated mast cell Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-a Resting Mast cell IL-4,

Journal of Leukocyte Biology 2003, 73 165-171

PMA/Ionomycin Induced
Constitutive & Induced Cytokine Expression by KU812 Basophils b-actin IL-3 IL-4 IL-5 IL-6 IL-8 IL-13 IFN-g 516bp Constitutive PMA/Ionomycin Induced

Der p1 Induces Cytokine Type-2 Cytokine mRNA
Expression in KU812 516bp IL-4 IL-5 IL-13 IFN-g Der p1 Inhibited Inhibitors +ve -ve b-actin

Protease Inhibitors Do Not Prevent Cytokine mRNA
Expression by KU812 516bp IL-13 516bp b-actin - - + + PMA/Ionomycin Inhibitors - - + +

Non-Proteolytic Antigens Do Not Induce Cytokine
mRNA Expression by KU812 516bp IL-13 516bp b-actin - - - Tetanus toxoid + + -ve - - - - + PMA/Ionomycin Time (hr) 1 1 4 4 4

Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils

Necator Americanus Proteases Induce Type-2 Cytokine
Expression by KU812 516bp IFN-g 516bp IL-5 516bp IL-4 516bp IL-13 516bp b-actin -ve +ve - Inhibitors + Inhibitors 0 ES 0 ES 100ng/ml ES 200ng/ml ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 1000ng/ml ES

Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils

The switch to IgE

Lebman & Coffman 1988 J Exp Med 168, 853-862

Switch regions Ca2 Ce Cg4 Cg2 Ca1 Cg1 Cg3 Cd Cm
Sg3 Sg1 Sa1 Sg2 Sg4 Se Sa2 Sm Cm Cd Cg3 VDJ Switch regions - repetitive regions of DNA that physically recombine Upstream of C regions The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. Switching is mechanistically similar to V(D)J recombination. Cg3 VDJ IgG3 produced. Switch from IgM

Switch recombination to IgE
A three signal process: Antigen – controls entire process Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells

B Y Th Th T cell help to B cells IL-4 and IL-13 Antigen CD40
CD40 Ligand

A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells

Soluble help via IL-4 or IL-13 from T helper cells
IL-4R IL-13R IL-13 IL-4 IL-4 IL-13 IL-4Ra gC IL-4Ra IL-13Ra1/2 JAK1 JAK1 JAK3 TYK1 TYK2 Stat-6 P P P P Stat-6 P Stat-6 P Stat-6 P Dimerised Stat-6 translocates to nucleus P

A three signal process: Antigen Soluble help via IL-4 or IL-13 from T helper cells Cognate help via CD40 L from T helper cells

Cognate help via CD40 L from T helper cells
Ligation promotes aggregation in lipid rafts CD40 2 3 5 6 TNF receptor associated factors IkB NF kB Uninhibited NFkB translocates to the nucleus IkB NF kB

Activation of the Ie promoter
Activation/cytokine responsive promoter Ce1 Ce2 Ce3 Ce4 Se Ie PU.1 NFkB BSAP C/EBP Stat6 Ie AP-1 Induced by IL-4/IL-13 and CD40 ligation BSAP – B cell specific activator protein. C/EBP CCAAT/enhancer binding protein. PU.1 – Spi1 equivalent in humans, ets transcription factor

Germline IgE transcripts
Transcription Ce1 Ce2 Ce3 Ce4 Se Ie Stat6 NFkB C/EBP PU.1 DNA Ce1 Ce2 Ce3 Ce4 Se Ie RNA Ce Ie Spliced RNA Germline transcripts Why has this mechanism evolved to transcribe just the C region? VHDHJH is needed to make a functional IgE Why is the epsilon switch region spliced out?

What do germline transcripts do?
Ce Ie Ce1 Ce2 Ce3 Ce4 Se RNA Spliced Se RNA Ce1 Ce2 Ce3 Ce4 Se Ie Stat6 NFkB C/EBP PU.1 S region RNA hybridises with template DNA

Mechanism of class switch recombination
Single stranded DNA 1. S region in the genomic DNA ‘melts’ Ie Ce1 Se Se 2. S region RNA spliced from germline RNA transcript hybridises to single-stranded DNA R loop 3. ssDNA R loop formed – a substrate for AID - ACTIVATION- INDUCED CYTIDINE DEAMINASE Se 5’ 3’

Activation-induced cytidine deaminase
B cell activation by antigen leads to: Soluble help via Th cell IL-4 or IL-13 Induces Stat 6 Cognate help via Th cell CD40 L from T helper Releases NFkB from IkB NFkB Stat6 Activation-induced cytidine deaminase gene AID gene is expressed under the same conditions as B cells induced to switch Ig isotype

Activation-induced cytidine deaminase
Expressed only in B cells Involved in isotype class switching & somatic hypermutation AID knockout mice do not class switch Ig isotype Ectopic expression in non B cells causes class switch Mutation in the AID gene can cause hyper IgM syndrome Deaminates cytidine on ssDNA, i.e. substitutes U for C

Preferred Se region target sequence for AID
GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA IgE S region and contains RGYW (A/G G T/C A/T) motifs Non-template strand is G-Rich AID RPA Replication protein A (RPA) targets AID to ssDNA in R loops by binding to RGYW motifs AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT

Activation induced cytidine deaminase
GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA Non-template ssDNA RNA/template DNA hybrid AID may also deaminate C on the template strand ?RNAase? NH2 N O Cytidine O N HN Uridine AID AID mediated deamination of cytidine to Uridine

GGGUTGA GGGUTGA CCCGACT CCCGACT G - U mismatch repair
S region DNA now contains mismatched G – U pairs that must be repaired e.g. by the base excision repair mechanism GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA Uracil-DNA glycolase (UNG) removes uracil to leave abasic sites in S region UNG GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA P GGGUTGA CCCGACT GGGUTGA CCCGACT Base is removed, but backbone remains intact

GGGUTGA CCCGACT GGGUTGA CCCGACT G - U mismatch repair
APE1 Abasic site is processed by the apurinic/apyrimidimic endonuclease 1 (APE1) GGGUTGA CCCGACT P OH GGGUTGA CCCGACT P DNA is now nicked to produce a single strand break GGGCTGGGU TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAU TCGAYTYNA Similar mechanism on the template strand creates a staggered double strand break GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA APE1

Processing of staggered ends
GGGCTGGG TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGA TCGAYTYNA GGGCTGGG CCCGACCCGACTCGACYCGACTCGACYCGA Exonuclease activity TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT TCGAYTYNA End fill-in reactions ACTCGACYCGACTCGACYCGAC Ca2 Ce Cg4 Cg2 Ca1 Cg1 Cg3 Cd Cm Sg3 Sg1 Sa1 Sg2 Sg4 Se Sa2 Sm

Process occurs in two S regions simultaneously
Activation of Im & Ie promoter by Ag, IL-4/13 and CD40L Production of germline transcripts and splicing of Sm and Se Deamination of ssDNA in Sm and Se by AID Base excision and mismatch repair Blunt-ended ds breaks and synapsis of Sm to Se by non-homologous end joining Ca2 Ce Cg4 Cg2 Ca1 Cg1 Cg3 Cd Cm Sg3 Sg1 Sa1 Sg2 Sg4 Se Sa2 Sm Cm Cd Cg3 Cg1 Ca1 Cg2 Cg4 Excised episomal circle of intervening DNA Cm Cd Cg3 VDJ Cg1 Ca1 Cg2 Cg4 Ce Ca2 VDJ Ce Ca2

Non-homologous end joining in class switch
Closely resembles another B cell Ig gene mechanism After N and P nucleotides have been inserted, several other proteins, (Ku70:Ku80, XRCC4 and DNA dependent protein kinases,ARTEMIS exonuclease, DNA ligase IV) bind to the hairpins and the heptamer ends. Ig gene recombination V D J 7 23 9 12 Defects in NHEJ proteins impair class switch

BCL-6 BCL-6 binds to the Stat-6 binding site and represses switching
BCL-6 -/- mice have enhanced IgE isotype switching BCL-6 -/- Stat6 -/- mice have no IgE An RFLP has been mapped to the first intron of the BCL-6 gene that is significantly associated with atopy - but not IgE levels Stat6 BCL-6 Transcription blocked Stat6 NFkB C/EBP PU.1 BSAP BCL-6 Stat6 is involved in Th2 cell differentiation, the expression of CD23 (the low affinity IgE receptor) and VCAM expression BCL-6 may exert it’s anti/pro-allergic activities via these genes

Additional areas to think about
Can’t get over a 2.2 mark without showing evidence of outside reading in answers

Relationship between isotype switch, somatic hypermutation and proliferation of B cells in the germinal centre What is the relationship between the deliberately mutagenic mechanisms of isotype switch and somatic hypermutation in B cells and the propensity of B cells to form tumours Where are the holes in the ‘skew to Th2’ model of allergy? What are allergic responses really for?

What are allergic immune responses really for?
Ascaris Enterobious Hookworm Leishmania Onchocerca Plasmodium Schistosome Taenia Toxoplasma Trichuris Trypanosoma Wuchereria

Text book view Helminth infections induce IgE, mastocytosis and eosinophilia A classic Th2-driven response Eosinophils killing a schistosome egg in vitro

Yet IL-4 may be involved - Trichuris muris model
However…….. Heavily parasitised individuals exist - despite Th2 responses and eosinophilia. Scarce in vivo evidence of eosinophil and IgE control of helminth infection Yet IL-4 may be involved - Trichuris muris model Susceptible mice Resistant mice Else et al., 1994 J. Exp Med

Th2 cells themselves may not be needed
Nippostrongylus infection IL-4 IL-4 from any source is sufficient to induce worm expulsion Urban et al., 1995 J. Immunol. 154,

Mechanisms of Allergic Immunity

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